This grant application responds to Program Announcement Number PA-11-110 (Ruth L. Kirschstein National Research Service Awards for Individual Predoctoral MD/PhD and Other Dual Doctoral Degree Fellows) by proposing to develop novel evolutionary platforms and apply computational modeling strategies to engineer soluble HIV gp140 trimers and epitope-scaffold mimetics with improved stability and accessibility of key functionally conserved quaternary broadly neutralizing antibody (bNAb) epitopes. These epitope-focused antigens are expected to elicit broadly neutralizing responses more strongly than native viral trimers, which evade immune recognition by providing limited access to functionally critical epitopes. Epitope-focused antigens may be used as vaccine candidates, baits for isolation of new bNAbs, and epitope probes to characterize serum antibody responses. To engineer epitope-focused trimers, a platform for yeast display of trimeric gp140 envelope will be developed and used to generate both randomly and rationally mutated libraries of trimers. These libraries will be evolved for enhanced binding to quaternary epitope-specific bNAbs to isolate epitope-focused candidate trimers. To engineer epitope-focused mimetics, a computational protein peeling strategy developed by collaborators will be applied to generate rewired epitope-scaffolds of quaternary epitopes. We will produce and test the designed epitope-scaffolds for expression, stability, aggregation propensity, and binding to conformational monoclonal bNAbs. The top-performing candidates will then be carried into immunogenicity studies in mice to determine whether elicited antibody responses target the intended epitopes and recognize natively folded trimers. By the end of the proposed funding period, we expect to have generated a widely applicable novel platform for directed evolution of multimeric proteins, an improved computational protein peeling method for generation of rewired epitope-scaffold mimetics informed by our results, and multiple quaternary epitope-focused vaccine candidates. These advances will serve the global community by offering both strategies towards and candidates for the development of HIV vaccines with improved elicitation of protective broadly neutralizing antibody responses.